Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths among malignancies. PDAC is highly invasive and forms metastases in distant organs at the very early stage of tumor progression. To better understand PDAC metastasis, tumor-blood vessel interactions need to be evaluated further, as tumor cells spread primarily through the blood circulation. However, how PDAC interacts with blood vessels and establishes distant metastases are poorly understood. Recently, our study using both three-dimensional (3D) biomimetic PDAC-on-chip and multiple in vivo mouse models showed that PDAC cells invaded blood vasculatures and actively replaced endothelial cells via ALK7 signaling, leading to a formation of tumor-vessel hybrid structure in PDAC tumors. We refer to this phenomenon as tumor vessel replacement. Despite the novelty of the finding, it is unknown what the biological consequences of the tumor vessel replacement in PDAC are. Understanding the phenotypic consequences of the tumor vessel replacement is critical to determine the clinical relevance and significance of blocking ALK7 in PDAC. We hypothesize that PDAC tumor vessel replacement increases tumor vessel permeability; then promotes tumor intravasation and metastasis by facilitating tumor cells’ entering the blood circulation through the leakier vessels. In order to test these hypotheses, we aim to determine—in both in vitro and in vivo—(i) if tumor vessel replacement induces tumor vessel leakiness and promotes metastatic dissemination and (ii) if ALK7 inhibition or ALK7 knock out (KO) ameliorates or reverses tumor vessel leakiness and metastasis. In Aim 1, we will assess the role of ALK7 in PDAC vessel permeability in pericyte-covered blood vessel on-chip by co-culturing microvascular endothelial cells and pericytes to mimic physiological blood vessels surrounded by pericytes (Aim 1.1). Next, we will evaluate the role of ALK7 in PDAC vessel dysfunction in vivo. We will generate an orthotopic PDAC model using wild-type or ALK7-KO PDAC cells, and examine PDAC tumor vessel permeability by intravenously injecting dextran molecules (Aim 1.2). In Aim 2, we will examine ALK7 in PDAC metastasis in vitro by establishing pre-metastatic liver microenvironment in the reservoirs that are connected to the engineered blood vessel. Multiple PDAC lines will be assessed to test whether ALK7-mediated tumor vessel replacement affects metastatic spreading (Aim 2.1). We will then evaluate the role of ALK7 in PDAC metastasis in vivo using human patient-derived xenograft (PDX) models in collaboration with Dr. Manuel Hidalgo. Metastatic tumor burdens in control vs. ALK7 KO groups will be assessed, and the number of circulating tumor cells and overall survival rate will be determined (Aim 2.2). In summary, our 3D PDAC-on-chip system will provide a unique platform to better investigate PDAC interactions with blood vessels and metastatic progression. We will decipher the roles of ALK7 signaling in mediating tumor vessel dysregulation and metastasis, and assess whether we will be able to reduce PDAC progression and metastasis by targeting ALK7.